The human microbiome is the collection of microorganisms that colonize the human body. Over the last year the analysis of the microbiome of 250 healthy subjects was initiated, focusing on five body sites (nasal, oral, vaginal, dermal, and gut) and many subsites. This study began to build a catalog of genome sequences of culturable bacteria that inhabit the human body as well as described the community structure formed by microbial species, allowing diversity to be assessed between body sites and between individuals. The goal of this study is to extend these initial probes in multiple directions. The study will add 1,000 genomes to the catalog of bacterial reference sequences of the human microbiome by using automated methods to isolate organisms and high throughput DNA sequencing to construct genome sequences. The study will also further characterize the metagenomes of the 250 individuals by directly comparing DNA sequences from organisms in the microbiome, thus deterimining what is common or variant between subjects. Going beyond bacteria, the project will characterize the virome, the component of the human microbiome formed by viruses, and take a census of eukaryotic microbes in the human microbiome. The last goal is to analyze the transcriptional patterns of microbes found in metagenomic communities, and by this means determine what functions are expressed in the different body niches. These studies will advance our growing knowledge of the human microbiome in healthy subjects, providing a baseline for disease studies as well as new tools for probing the microbiome. Public Health Relevance: The human microbiome is the collection of microorganisms that colonize the human body. Over the last year the analysis of the microbiome of 250 healthy subjects was initiated, focusing on five body sites. This study began to build a catalog of genome sequences of organisms that inhabit the human body as well as described the diversity found between body sites and individuals. The goal of this study is to add 1,000 genomes to the catalog of reference sequences of the human microbiome, to further characterize differences in the metagenomes of 250 individuals by directly comparing shotgun sequences, to characterize the virome component of the human microbiome, to take a census of eukaryotic microbes in the human microbiome, and to analyze the transcriptional pattern found in metagenomic communities. These studies will advance our growing knowledge of the human microbiome in healthy subjects, providing a baseline for disease studies as well as new tools for probing the microbiome.